Reversal of drug resistance mediated by multidrug resistance protein (MRP)1 by dual effects of agosterol A on MRP1 function

We previously isolated agosterol A (AG-A) from a marine Spongio sp. and fou nd that it completely reversed colchicine resistance in P-glycoprotein (Pgp )-over-expressing KB-C2 cells and vincristine resistance in multidrug-resis tance protein (MRP)I -over-expressing CV60 cells. However, a tri-deacetylat ed derivative of AG-A (IAG-A) showed almost no activity in reversing Pgp- o r MRP I-mediated drug resistance. In this study, we examined the mechanisms by which AG-A reverses MRP mediated drug resistance by investigating the i nteraction between agosterols and MRPI in MRPI-over-expressing human KB car cinoma (KB/MRP) cells. [H-3]-Leukotriene C, (LTC,), [H-3]-2,4-dinitrophenyl -S-glutathione uptake into membrane vesicles prepared from KB/MRP cells and intracellular [H-3]-vincristine accumulation and efflux in KB/MRP cells we re measured with or without AG-A and/or inactive IAG-A. AG-A reduced MRPI-m ediated [H-3]-LTC4 transport in a dose-dependent manner, but IAG-A did not. Inhibition by AG-A was competitive, with a K,value of 31 muM. AG-A at 10 m uM enhanced the accumulation of [H-3]-vincristine in KB/MRP cells to the le vel of that in control cells in the absence of the agent. Likewise, ATP-dep endent efflux of [H-3]-vincristine from KB/MRP cells was enhanced compared with KB-3-I cells and inhibited by AG-A. In addition, AG-A reduced intracel lular levels of glutathione, a compound required for MRPI-mediated transpor t of some anti-cancer drugs. These findings suggest that AG-A reverses MRPI -mediated drug resistance by directly inhibiting the capacity of MRPI to tr ansport drugs. In addition, the capacity of AG-A to reduce cellular glutath ione levels may contribute to the modulating activity of MRPI. (C) 2001 Wil ey-Liss, Inc.